Synthesis, photochemical synthesis and antitumor evaluation of novel derivatives of thieno[3',2':4,5]thieno[2,3-c]quinolones.

Article abstract of DOI:10.1248/cpb.50.656

The novel derivatives of thieno[3',2':4,5]thieno[2,3-c]quinolones 6a, 6b, 7, 10a and 10b were synthesized in multistep synthesis starting from thiophene-3-carboxaldehyde and malonic acid reacting in aldol condensation or from 3-bromothiophenes or methyl 4-bromothiophene-2-carboxylate reacting in Heck reaction. They resulted in corresponding substituted thienylacrylic acids 3a-c, which were cyclized into thieno[2,3-c]thiophene-2-carbonyl chlorides 4a-c and converted into thieno[2,3-c]thiophene-2-carboxamides 5a-d. Prepared carboxamides were photochemically dehydrohalogenated into corresponding substituted thieno[3',2':4,5]thieno[2,3-c]quinolones 6a-d. Compound 7 was prepared from 6d by alkylation with N-[3-(dimethylamino)propyl]chloride hydrochloride in the presence of NaH. Compounds 10a and 10b were prepared from 6c in the multistep synthesis over acid 8 and acid chloride 9. Compounds 6a, 6b, 7, 10a and 10b were found to exert cytostatic activities against malignant cell lines: pancreatic carcinoma (MiaPaCa2), breast carcinoma (MCF7), cervical carcinoma (HeLa), laryngeal carcinoma (Hep2), colon carcinoma (CaCo-2), melanoma (HBL), and human fibroblast cell lines (WI-38). The compound 6b, which bears the 3-dimethylaminopropyl substituent on quinolone nitrogen and methoxycarbonyl substituent on position 9, exhibited marked antitumor activity. On the contrary, compound 7, which also bears the 3-dimethylaminopropyl substituent on the quinolone nitrogen but anilido substituent on position 9, exhibited less antitumor activity than the others.

Full text of DOI:10.1248/cpb.50.656

656
Notes
Chem. Pharm. Bull. 50(5) 656—660 (2002)
Vol. 50, No. 5
Synthesis, Photochemical Synthesis and Antitumor Evaluation of Novel
Derivatives of Thieno[3؅,2؅:4,5]thieno[2,3-c]quinolones
Jasna DOGANKORUZˇNJAK,a,1) Neda SLADE,b Branimir ZAMOLA,c Kresˇimir PAVELIC´,b and
,a
*
Grace KARMINSKI-ZAMOLA
a Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb; Marulic´ev trg
20, P. O. Box 177, HR-10000 Zagreb, Croatia: b Division of Molecular Medicine, Ruder Bosˇkovic´ Institute; Bijenicˇka cesta
¯
54, P. O. Box 1016, HR-10000 Zagreb, Croatia: and c Department of Biochemical Engineering, Faculty of Food and
Biotechnology, University of Zagreb; Pierottieva 6, HR-10000 Zagreb, Croatia.
Received November 9, 2001; accepted January 15, 2002
The novel derivatives of thieno[3؅,2؅:4,5]thieno[2,3-c]quinolones 6a, 6b, 7, 10a and 10b were synthesized in
multistep synthesis starting from thiophene-3-carboxaldehyde and malonic acid reacting in aldol condensation
or from 3-bromothiophenes or methyl 4-bromothiophene-2-carboxylate reacting in Heck reaction. They resulted
in corresponding substituted thienylacrylic acids 3a—c, which were cyclized into thieno[2,3-c]thiophene-2-car-
bonyl chlorides 4a—c and converted into thieno[2,3-c]thiophene-2-carboxamides 5a—d. Prepared carboxamides
were photochemically dehydrohalogenated into corresponding substituted thieno[3؅,2؅:4,5]thieno[2,3-c]-
quinolones 6a—d. Compound 7 was prepared from 6d by alkylation with N-[3-(dimethylamino)propyl]chloride
hydrochloride in the presence of NaH. Compounds 10a and 10b were prepared from 6c in the multistep synthesis
over acid 8 and acid chloride 9. Compounds 6a, 6b, 7, 10a and 10b were found to exert cytostatic activities
against malignant cell lines: pancreatic carcinoma (MiaPaCa2), breast carcinoma (MCF7), cervical carcinoma
(HeLa), laryngeal carcinoma (Hep2), colon carcinoma (CaCo-2), melanoma (HBL), and human fibroblast cell
lines (WI-38). The compound 6b, which bears the 3-dimethylaminopropyl substituent on quinolone nitrogen and
methoxycarbonyl substituent on position 9, exhibiedt marked antitumor activity. On the contrary, compound 7,
which also bears the 3-dimethylaminopropyl substituent on the quinolone nitrogen but anilido substituent on po-
sition 9, exhibited less antitumor activity than the others.
Key words quinolone; thiophene; antitumor activity; Heck reaction; photocyclization; alkylation
There is little literature data describing the antitumor ac- Results and Discussion
tivity of heterocyclic quinolones. Some new analogs of 4-
Chemistry Compounds 6a and 6b were prepared in
quinolinones and 1,7 naphthyridin-4-ones were synthesized multistep synthesis according to Chart 1. Starting from thio-
and their in vitro antitumor activity against central nervous phene-3-carboxaldehyde and malonic acid by already known
system (CNS) (SNB-75), breast (T-47D) and lung (NCI-H aldol condensation reaction,16) or from 3-bromothiophene by
522) cancer cell lines was tested.2) One of the first quinoline- Heck reaction,17) or from 4-bromothiophene-2-carboxalde-
quinones, streptonigrin (SN) is an antitumor antibiotic, hyde by oxidation of carboxaldehyde group18) and esterifica-
which has activity against a broad range of tumors.3—5) SN tion of carboxylic acid,19) followed by Heck reaction, 5-sub-
was studied clinically as an antitumor agent, but its use was stituted 3-thienyl-acrylic acids 3a—c were prepared. Cyclisa-
limited because of reports of delayed myelotoxicity.6,7) Nev- tion of 3a—c with thionyl chloride and a catalytic amount of
ertheless, positive results were reported for SN either as a pyridine, by the known method20,21) gave substituted 3-
single agent8,9) or in combination chemotherap,10,11) SN is an chlorothieno[2,3-b]thiophene-2-carbonyl chlorides 4a—c.
excellent substrate for oxidoreductase (NQ01).12)
By refluxing of chlorides 4b and 4c with aniline in toluene
Pyranoquinoline-2-ones were synthesized and evaluated corresponding 3-chlorothieno[2,3-b]thiophene-2-carboxam-
for their in vitro cytotoxicity against a panel of human tumor ides 5c and 5d were obtained.
cell lines,13) while 2-arylquinazolinones displayed significant
On the other hand, corresponding substituted 3-chloroth-
growth inhibitory action against tumor cell lines and some of ieno[2,3-b]thiophene-2-carboxamides 5a and 5b were ob-
them were potent inhibitor of tubulin polymerization. Some tained from chlorides 4a—c by the Shotten–Baumann
of them displayed selective activity against P-gp-expressing method22) with N-[3-(dimethyamino)propyl]aniline at 0 °C.
epidermoid carcinoma of the nasopharynx.14) Recently triflu-
All prepared anilides 5a—d were converted by photo-
oromethyl substituted pyranoquinolinone was tested for its chemical dehydrohalogenation reaction into corresponding
ability to modulate the transcriptional activity of the human quinolones 6a—d.20) Better yields were achieved in cases
androgen receptor (HAR).15)
where N-atom on the anilide part of the molecule was not
Searching for compounds related to these classes of bio- substituted with N-[3-(dimethyamino)propyl] substituent.
logically and pharmacologically active compounds, we pre- Quinolone 7 was obtained by alkylation of 6d with N-[3-(di-
pared new substituted thieno[3Ј,2Ј:4,5]thieno[2,3-c]- methyamino)propyl]chloride hydrochloride in the presence
quinolones containing N-[3-(dimethyamino)propyl] sub- of NaH/N,N-dimethylformamide (DMF).23) Hydrolysis of
stituent on the quinolone nitrogen (6a, 6b, and 7) and those ester group in position 9 of the quinolone 6c gave acid 8
containing N-[3-(dimethyamino)propyl] substituent in the which, in the reaction with SOCl2, gave acid chloride 9,
amide part of the molecule (10a and 10b).
which was then converted into the quinolones 10a in the re-
action with N-[3-(dimethyamino)propyl]amine and 10b in
To whom correspondence should be addressed. e-mail: gzamola@pierre.fkit.hr
© 2002 Pharmaceutical Society of Japan

May 2002
657
Chart 1
the reaction with N-[3-(dimethyamino)propyl]aniline.24) Com-
pound 10a was also prepared directly from quinolone 6c in
the reaction with N-[3-(dimethyamino)propyl]amine.25)
The structures of the novel compounds were determined
on the basis of analysis of chemical shifts and H–H coupling
constants in 1H- and 13C-NMR spectra as well as by elemen-
tal analysis.
Antitumor Activity The effect of all compounds on cell
proliferation was tested on the following malignant human
tumor cell lines: cervical carcinoma (HeLa), breast carci-
noma (MCF-7), colon carcinoma (CaCo-2), pancreatic carci-
noma (Mia PaCa-2), melanoma (HBL) and laryngeal carci-
noma (Hep-2), as well as on human fibroblast cell line (WI-
38).
Inhibitory effects are shown in Table 1 and Fig. 1. All
tested compounds exhibited strong antitumor activity with
IC50 ranging from 0.1 to 51 mM. Only compound 7 showed
weaker inhibition than the others, IC50 ranging from 4.6 mM
for HBL to 51 mM for Hep-2 cells. Compounds 6a and 10a
were the strongest inhibitors of growth for HeLa cells

658
Vol. 50, No. 5
Table 1. Inhibitory Effects of Compounds 6a, 6b, 7, 10a, and 10b on the was performed at room temperature with a water cooled immersion well fit-
Growth of Tumor Cell Lines and Normal Fibroblasts (WI-38)
ted with an “Original Hanau” 400 W high pressure mercury arc lamp using a
quartz or pyrex filter. All compounds were routinely checked by TLC with
Merck silica gel 60F-254 glass plates or Merck aluminium oxide plates.
3-(3-Thienyl)acrylic Acid (3a) Method A: A solution of thiophene-3-
carboxaldehyde (10 ml, 0.11 mol), malonic acid (11.40 g, 0.11 mol) and
piperidine (1.5 ml) in pyridine (100 ml) was refluxed for 18 h. After cooling
to room temperature the reaction mixture was poured into ice-water (ca.
300 g) and acidified with concentrated HCl with vigorous stirring. The pre-
cipitated crystals were filtered, washed with water and crystallized from 50%
ethanol to give 3a (10.17 g, 60.0%) as white crystals, mp 150—152 °C (lit.16)
mp 151 °C).
IC50 (mM)a)
Compd.
HeLa
MCF-7 CaCo-2 Mia PaCa-2 HBL
Hep-2
WI-38
6a
6b
7
10a
10b
0.1
2.2
7.41
6.23
0.23
1.0
4.57
1.0
0.46
6.17
4.5
5.82
2.5
2.88
4.67
7.59
4.9
31.6
0.1
47.9
38.0
34.7
51.0
1.0
2.5
6.3
0.15
3.8
0.74
2.34
7.24
0.59
2.5
4.17
5.76
Method B17): A mixture of 3-bromothiophene (3 ml, 0.03 mol), cyclohexyl-
ammonium acrylate (20 g, 0.12 mol), palladium(II)acetate (0.10 g, 0.45
mmol), triphenylphosphine (0.36 g, 1.37 mmol), triethylamine (30 ml) and
acetonitrile (70 ml) was sealed in a glass tube and heated at 120 °C for 20 h.
After evaporation of the solvent, the residue was dissolved in water and
boiled with charcoal. After filtration, the filtrate was acidified with diluted
HCl (1 : 1). The resulting precipitate was filtered off and recrystallised from
50% ethanol to give 3a (1.25 g, 25.3%) as white crystals.
a) 50% inhibitory concentration, or compound concentration required to inhibit cell
proliferation by 50%.
3-(5-Methoxycarbonyl-3-thienyl)acrylic Acid (3b) Compound 3b was
synthesised according to method B starting from methyl 4-bromothiophene-
2-carboxylate (2) in 70.2% yield, mp 197—200 °C. IR (KBr) cmϪ1: 1725
1
(CϭO), 1681 (CϭO), 1630 (CϭC). H-NMR (DMSO-d6) d: 12.86 (1H, s),
8.23 (1H, d, Jϭ1.25 Hz), 8.10 (1H, d, Jϭ1.24 Hz), 7.57 (1H, d, Jϭ
15.88 Hz), 6.47 (1H, d, Jϭ15.87 Hz), 3.84 (3H, s). 13C-NMR (DMSO-d6) d:
168.0 (s), 163.0 (s), 138.2 (s), 137.5 (d), 136.2 (s), 134.8 (d), 131.8 (d),
120.2 (d), 52.6 (q). MS m/z: 213 (Mϩ1). Anal. Calcd for C9H8O4S: C,
50.94; H, 3.80. Found: C, 49.28; H, 3.57.
3-(5-Carboxy-3-thienyl)acrylic Acid (3c) Compound 3c was synthe-
sized according to method B starting from 4-bromothiophene-2-carboxylic
acid (1) in 84.7% yield, mp 264—266 °C (lit.20) mp 264—266 °C).
3-Chlorothieno[2,3-b]thiophene-2-carbonyl Chloride (4a) The solu-
tion of acid 3a (10 g, 0.07 mol), thionyl chloride (50 ml) and pyridine (2 ml)
in chlorobenzene (100 ml) was heated at 140 °C for 18 h. Excess of thionyl
chloride was removed under reduced pressure and the residue extracted with
boiling cyclohexane. After cooling, the precipitated yellow crystals were
filtered off to give 4a (3.23 g, 40.5%), mp 127—130 °C (lit.21) mp 127—
130 °C).
Fig. 1. The Inhibitory Effect of Different Concentrations of One of the
Most Active Compounds (6a) on the Growth of Human Malignant Tumor
Cell Lines (HeLa, MCF-7, CaCo-2, Mia PaCa-2, HBL, Hep-2) and Normal
Human Fibroblasts (WI-38)
(IC50ϭ0.1 mM) and for CaCo-2 cells (IC50ϭ0.23 and 0.15 mM,
respectively). 6a and 10b were also strong inhibitors of the
growth of HBL cells (IC50ϭ0.46 and 0.59 mM). Compounds
6a and 10a showed the most pronounced selectivity in cyto-
static activity—IC50 from 0.1 mM for HeLa cells to 7.41 and
7.24 mM for MCF-7 and HBL, respectively.
Methyl 3-Chloro-2-chlorocarbonylthieno[2,3-b]thiophene-5-carboxyl-
ate (4b) Starting from acid 3b using the same procedure as above, com-
pound 4b was obtained as yellow crystals in 56.6% yield, mp 135—138 °C.
1
IR (KBr) cmϪ1: 1771 (CϭO), 1727 (CϭO). H-NMR (CDCl3) d: 8.24 (1H,
s), 4.00 (3H, s). 13C-NMR (CDCl3) d: 163.8 (s), 161.8 (s), 141.2 (s), 136.9
(s), 134.7 (d), 134.1 (s), 129.8 (s), 124.6 (s), 52.7 (q). MS m/z: 295 (Mϩ1).
In comparison with antitumor activity of other quino- Anal. Calcd for C9H4Cl2O3S2: C, 36.62; H, 1.37. Found: C, 36.60; H, 1.34.
lones2,26—30) the substances tested in these experiments are
very strong inhibitors of tumor cell proliferation at very
small concentrations.
3-Chlorothieno[2,3-b]thiophene-2,5-dicarbonyl Dichloride (4c) Start-
ing from acid 3c using the same procedure as above, compound 4c was ob-
tained as yellow crystals in 42.0% yield, mp 156—160 °C (lit.20) mp 156—
161 °C).
N-[3-(Dimethylamino)propyl]-3-chlorothieno[2,3-b]thiophene-2-car-
boxanilide (5a) A solution of 4a (4.40 g, 0.02 mol) in chloroform (250 ml)
was added dropwise to a cold mixture of N-[3-(dimethylamino)propyl]ani-
line (3.56 g, 0.02 mol) and 5% NaOH (9 ml) at 2—3 °C. The resulting mix-
ture was stirred at the same temperature for 30 min and then for 1 h at room
temperature. The organic layer was separated and washed first with 10%
HCl, then with water and dried over anhydrous Na2SO4. After evaporation of
the solvent, the crude product was purified by column chromatography (neu-
tral aluminium oxide) with toluene–ethanol mixture (9 : 1, v/v) to yield
6.44 g (89.5%) of compound 5a. IR (NaCl) cmϪ1: 2767—2944 (CH2), 1634
Conclusions
All tested compounds exhibited strong inhibitory activities
against all cell lines tested. The cell sensitivity varied with
the compound applied. All compounds examined were of
similar cytotoxicity, except compound 7 which exhibited a
less cytotoxic effect against all cell lines.
Experimental
1
Chemistry Melting points were determined on a Kofler hot stage micro-
(CϭO). UV lmax (methanol) nm: 230, 288, 346. H-NMR (DMSO-d6) d:
scope and are uncorrected. IR spectra were recorded on a Nicolet Magna 7.67 (1H, d, Jϭ5.29 Hz), 7.37—7.21 (5H, m), 7.15 (1H, d, Jϭ5.32 Hz), 3.89
760 spectrophotometer in KBr discs or as a liquid film between sodium (2H, t, Jϭ7.37 Hz), 2.38 (2H, t, Jϭ7.12 Hz), 2.18 (6H, s), 1.74 (2H, tt,
chloride plates. UV spectra were recorded on either a Perkin-Elmer 124 or a Jϭ7.16, 7.39 Hz). 13C-NMR (DMSO-d6) d: 161.3 (s), 142.5 (s), 141.6 (s),
Hewlett-Packard 8452A spectrophotometer-meter in methanol or ethanol. 137.4 (s), 133.6 (s), 131.7 (d), 129.2 (d), 129.2 (d), 127.6 (d), 127.6 (d),
1H- and 13C-NMR spectra were recorded on either a Varian Gemini 300 or a 127.5 (d), 118.5 (d), 116.2 (s), 56.1 (t), 48.0 (t), 44.8 (q), 44.8 (q), 24.8 (t).
Bruker Avance DPX 300 spectrometer in DMSO-d6 or CDCl3. Chemical MS m/z: 379 (Mϩ1). Anal. Calcd for C18H19ClN2OS2: C, 57.05; H, 5.05; N,
shifts (d) are reported in parts per million (ppm) relative to tetramethylsilane 7.39. Found: C, 56.99; H, 5.10; N, 7.45.
(TMS) as an internal standard, and coupling constants (J) are given in hertz
Methyl 2-{N-[3-(Dimethylamino)propyl]-N-phenylcarbamoyl}-3-
(Hz). Mass spectra were recorded on either a Varian-MAT 311A (Electron chlorothieno[2,3-b]thiophene-5-carboxylate (5b) Starting from com-
Impact and Fast Atom Bombardment) or a Micromass Platform LCZ (Elec- pound 4b using the same procedure as above, compound 5b was obtained,
trospray). Elemental analysis for carbon, hydrogen and nitrogen was per- after crystallisation from ethanol, as white crystals in 37.1% yield, mp
formed on a Perkin-Elmer 2400 elemental analyser. Elemental compositions 134—135 °C. IR (KBr) cmϪ1: 2721—2946 (CH2), 1716 (CϭO), 1630
1
of the compounds agreed to within 0.4% of the calculated value. Irradiation (CϭO). UV lmax (methanol) nm: 208, 270. H-NMR (DMSO-d6) d: 7.75

May 2002
659
(1H, s), 7.33—7.28 (4H, m), 7.23 (1H, dd, Jϭ7.01 Hz), 3.87 (2H, t,
Jϭ7.31 Hz), 3.83 (3H, s), 2.25 (2H, t, Jϭ6.97 Hz), 2.08 (6H, s), 1.67 (2H, tt,
Jϭ7.31, 7.07 Hz). 13C-NMR (DMSO-d6) d: 162.4 (s), 161.6 (s), 143.6 (s),
142.3 (s), 142.2 (s), 137.6 (s), 136.0 (s), 130.0 (d), 130.0 (d), 128.4 (d),
128.4 (d), 128.4 (d), 125.0 (d), 117.4 (s), 57.1 (t), 53.4 (q), 49.0 (t), 45.9 (q),
45.9 (q), 26.0 (t). MS m/z: 437 (Mϩ1). Anal. Calcd for C20H21ClN2O3S2: C,
54.97; H, 4.84; N, 6.41. Found: C, 54.94; H, 4.80; N, 6.40.
above, after 2 h of irradiation followed by recrystallization from dioxane
compound 6d (0.17 g, 40.0%) was obtained as light yellow crystals, mp
Ͼ300 °C (lit.20) mp Ͼ300 °C).
5-(3-(Dimethylamino)propyl(-6-oxo-5,6-dihydrothieno[3؅,2؅:4,5]-
thieno[2,3-c]quinoline-9-carboxanilide Hydrochloride (7) To a cold so-
lution of 6d (1.00 g, 2.7 mmol) in anhydrous DMF (200 ml), sodium hydride
as 60—65% oil dispersion (0.50 g, 13.0 mmol) was added in three por-
tions.31) After stirring under nitrogen for 15 min, a solution of 3-(dimethyl-
amino)propyl chloride hydrochloride (1.12 g, 7.0 mmol) in DMF (50 ml) was
added to the solution and the reaction mixture was stirred for 2 h at 0 °C and
60 h at room temperature. The solid (sodium chloride) was separated by fil-
tration and the solvent was removed under reduced pressure. The crude
product was purified by column chromatography (neutral aluminium oxide)
with toluene–ethanol mixture (9 : 1, v/v) followed by stirring the solid with
saturated ethanolic HCl (10 ml) for 15 h to give 7 (0.12 g, 9.1%) as white
crystals, mp 238—240 °C. IR (KBr) cmϪ1: 2670—3059 (CH2), 1660
(CϭO), 1651 (CϭO). UV lmax (ethanol) nm: 230, 260, 328. 1H-NMR
(DMSO-d6) d: 10.59 (1H, s), 9.84 (1H, s), 7.57—7.37 (5H, m), 7.22 (1H, d,
Jϭ8.22 Hz), 7.05—6.92 (3H, m), 6.69 (1H, s), 3.10 (2H, t, Jϭ7.20 Hz), 2.94
(2H, t, Jϭ7.70 Hz), 2.55 (6H, s), 1.90 (2H, tt, Jϭ7.40 Hz). 13C-NMR
(DMSO-d6) d: 161.3 (s), 156.5 (s), 146.1 (s), 142.9 (s), 140.1 (s), 139.3 (s),
137.8 (s), 134.0 (s), 131.3 (d), 131.3 (d), 131.1 (s), 129.1 (d), 125.5 (d),
123.9 (d), 123.9 (d), 122.9 (d), 122.5 (d), 122.2 (d), 118.4 (s), 116.0 (d),
55.2 (t), 42.1 (q), 42.1 (q), 33.0 (t), 23.7 (t). MS m/z: 462 (Mϩ1; free base).
Anal. Calcd for C25H24ClN3O2S2: C, 60.29; H, 4.86; N, 8.44. Found: C,
60.14; H, 5.04; N, 8.42.
6-Oxo-5,6-dihydrothieno[3؅,2؅:4,5]thieno[2,3-c]quinoline-9-carboxylic
Acid (8) A solution of NaOH (0.60 g, 15.0 mmol) in water (100 ml) was
added to the solution of 7 (0.45 g, 1.4 mmol) in ethanol (50 ml). The result-
ing solution was refluxed for 1 h. Ethanol was removed under reduced pres-
sure, the residue was dissolved in water and acidified with 5% HCl. The pre-
cipitate was filtered off, washed with water and recrystallized from
DMSO–water mixture (1 : 2, v/v) to give 8 (0.32 g, 74.4%) as white crystals,
mp Ͼ300 °C. IR (KBr) cmϪ1: 1677 (CϭO), 1631 (CϭO). UV lmax
(methanol) nm: 226, 278, 316, 330, 346. 1H-NMR (DMSO-d6) d: 13.60 (1H,
s), 12.18 (1H, s), 8.76 (1H, s), 8.53 (1H, d, Jϭ7.88 Hz), 7.60 (1H, dd,
Jϭ7.18 Hz), 7.53 (1H, d, Jϭ7.18 Hz), 7.39 (1H, m, Jϭ7.46, 1.22 Hz). 13C-
NMR (DMSO-d6) d: 163.0 (s), 157.6 (s), 140.8 (s), 139.3 (s), 137.5 (s),
137.5 (s), 135.3 (s), 135.3 (s), 129.5 (d), 126.8 (d), 124.3 (d), 122.8 (d),
116.5 (d), 116.1 (s). MS m/z: 302 (Mϩ1). Anal. Calcd for C14H7NO3S2: C,
55.80; H, 2.34; N, 4.65. Found: C, 55.60; H, 2.11; N, 4.54.
Methyl 2-[N-Phenylcarbamoyl]-3-chlorothieno[2,3-b]thiophene-5-car-
boxylate (5c) A solution of 4b (2.55 g, 8.6 mmol) and aniline (1 ml,
11.0 mmol) in toluene (50 ml) was refluxed for 3 h. After cooling, precipi-
tated crystals were filtered off and recrystallised from acetone to give 5c
(1.81 g, 59.6%) as pale yellow crystals, mp 213—216 °C. IR (KBr) cmϪ1
:
1
1716 (CϭO), 1651 (CϭO). UV lmax (methanol) nm: 206, 270. H-NMR
(DMSO-d6) d: 10.34 (1H, s), 7.98 (1H, s), 7.69 (2H, d, Jϭ7.65 Hz), 7.38
(2H, dd, Jϭ7.66 Hz), 7.15 (1H, dd, Jϭ7.38 Hz), 3.89 (3H, s). 13C-NMR
(DMSO-d6) d: 161.6 (s), 158.6 (s), 143.0 (s), 142.6 (s), 138.0 (s), 136.9 (s),
135.7 (s), 128.9 (d), 128.9 (d), 124.7 (d), 124.5 (d), 120.3 (d), 120.3 (d),
117.5 (s), 52.8 (q). MS m/z: 352 (Mϩ1). Anal. Calcd for C15H10ClNO3S2: C,
51.21; H, 2.86; N, 3.98. Found: C, 51.06; H, 2.86; N, 3.86.
3-Chlorothieno[2,3-b]thiophene-2,5-dicarboxanilide (5d) Starting
from compound 4c using the same procedure as above, compound 5d was
obtained, after crystallization from DMF–water mixture (1 : 2, v/v), as pale
yellow crystals in 33.0% yield, mp 236—238 °C (lit.20) mp 236—238 °C).
5-[3-(Dimethylamino)propyl]-6-oxo-5,6-dihydrothieno[3؅,2؅:4,5]-
thieno[2,3-c]quinolin-6-one Hydrochloride (6a) A solution of anilide 5a
(0.65 g, 1.7 mmol) and triethylamine (0.23 ml, 1.7 mmol) in a methanol–ben-
zene mixture (1 : 10, v/v) (550 ml) was irradiated with a 400 W high pressure
mercury arc lamp using a pyrex filter at room temperature for 30 min. Dur-
ing the irradiation the air was bubbled through the solution. After evapora-
tion of the solvent, the crude product was purified by column chromatogra-
phy (neutral aluminium oxide) with toluene–ethanol mixture (9 : 1, v/v). The
oily residue was stirred with saturated ethanolic HCl (10 ml) for 15 h to give
6a (0.12 g, 31.3%) as white crystals, mp 260—263 °C. IR (KBr) cmϪ1
:
2474—3049 (CH2), 1634 (CϭO). UV lmax (ethanol) nm: 228, 238, 254,
1
270, 294, 306, 332, 346. H-NMR (DMSO-d6) d: 10.15 (1H, s), 8.53 (1H,
dd, Jϭ7.94, 1.00 Hz), 8.16 (1H, d, Jϭ5.45 Hz), 7.92 (1H, d, Jϭ5.42 Hz),
7.82 (1H, d, Jϭ8.47 Hz), 7.68 (1H, m, Jϭ8.55, 7.18, 1.32 Hz), 7.46 (1H, dd,
Jϭ7.26, 7.77 Hz), 4.50 (2H, t, Jϭ7.25 Hz), 3.23 (2H, t, Jϭ7.89 Hz), 2.74
(6H, s), 2.19 (2H, tt, Jϭ7.56 Hz). 13C-NMR (DMSO-d6) d: 157.2 (s), 143.7
(s), 141.2 (s), 136.9 (s), 133.8 (s), 133.0 (s), 131.8 (d), 129.5 (d), 124.8 (d),
122.7 (d), 121.3 (d), 117.5 (s), 115.7 (d), 53.9 (t), 41.9 (q), 41.9 (q), 39.2 (t),
22.6 (t). MS m/z: 343 (Mϩ1; free base). Anal. Calcd for C18H19ClN2OS2: C,
57.05; H, 5.05; N, 7.39. Found: C, 57.29; H, 5.21; N, 7.24.
6-Oxo-5,6-dihydrothieno[3؅,2؅:4,5]thieno[2,3-c]quinoline-9-carbonyl
Chloride (9) A mixture of 8 (0.23 g, 0.8 mmol) and thionyl chloride (5 ml,
68.8 mmol) was refluxed for 4 h. Excess of thionyl chloride was removed
under reduced pressure to give 9 (0.24 g, 100%) as yellow crystals, mp
Methyl 5-(3-(Dimethylamino)propyl(-6-oxo-5,6-dihydrothieno-
[3؅,2؅:4,5]thieno[2,3-c]quinoline-9-carboxylate Hydrochloride (6b)
Starting from 5b (0.50 g, 1.1 mmol) and using the same procedure as above,
after 15 min of irradiation followed by stirring with saturated ethanolic HCl
(10 ml) compound 6b (0.38 g, 75.4%) was obtained as light yellow crystals,
mp 240—242 °C. IR (KBr) cmϪ1: 2472—2948 (CH2), 1705 (CϭO), 1634
(CϭO). UV lmax (ethanol) nm: 228, 242, 282, 334, 348. 1H-NMR (DMSO-
d6) d: 8.75 (1H, s), 8.55 (1H, d, Jϭ8.00 Hz), 7.82 (1H, d, Jϭ8.65 Hz), 7.70
(1H, dd, Jϭ7.33, 8.32 Hz), 7.45 (1H, dd, Jϭ7.33, 7.63 Hz), 4.47 (2H, t,
Jϭ6.94 Hz), 3.93 (3H, s), 3.39 (2H, dt, Jϭ5.05, 7.97 Hz), 3.23 (2H, tt,
Jϭ7.22 Hz), 2.76 (3H, s), 2.74 (3H, s). 13C-NMR (DMSO-d6) d: 161.8 (s),
157.1 (s), 149.4 (s), 140.3 (s), 137.1 (s), 137.1 (s), 134.4 (s), 133.8 (s), 130.0
(d), 127.4 (d), 125.2 (d), 123.1 (d), 117.3 (s), 115.9 (d), 54.1 (t), 52.7 (q),
42.1 (q), 42.1 (q), 39.3 (t), 22.8 (t). MS m/z: 401 (Mϩ1; free base). Anal.
Calcd for C20H21ClN2O3S2: C, 54.97; H, 4.84; N, 6.41. Found: C, 54.91; H,
4.90; N, 6.39.
Methyl 6-Oxo-5,6-Dihydrothieno[3؅,2؅:4,5]thieno[2,3-c]quinoline-9-
carboxylate (6c) Starting from 5c (0.53 g, 1.5 mmol) and using the same
procedure as above, after 30 min of irradiation followed by recrystallization
from DMF compound 6c (0.36 g, 75.8%) was obtained as light brown crys-
tals, mp Ͼ300 °C. IR (KBr) cmϪ1: 1728 (CϭO), 1667 (CϭO). UV lmax
(methanol) nm: 208, 226, 240, 282, 332, 346. 1H-NMR (DMSO-d6) d: 12.20
(1H, s), 8.85 (1H, s), 8.55 (1H, d, Jϭ7.85 Hz), 7.61 (1H, m, Jϭ8.17, 7.02,
1.06 Hz), 7.53 (1H, dd, Jϭ8.18, 1.19 Hz), 7.40 (1H, m, Jϭ7.48, 1.31 Hz),
3.95 (3H, s). 13C-NMR (DMSO-d6) d: 161.9 (s), 157.6 (s), 140.7 (s), 137.5
(s), 137.5 (s), 137.1 (s), 135.3 (s), 134.8 (s), 129.5 (d), 127.4 (d), 124.4 (d),
122.9 (d), 116.5 (d), 116.1 (s), 52.7 (q). MS m/z: 316 (Mϩ1). Anal. Calcd
for C15H9NO3S2: C, 57.13; H, 2.88; N, 4.44. Found: C, 57.10; H, 2.98; N,
4.70.
1
Ͼ300 °C. IR (KBr) cmϪ1: 1741 (CϭO), 1661 (CϭO). H-NMR (CDCl3) d:
12.18 (1H, s), 8.86 (1H, s), 8.53 (1H, d, Jϭ7.90 Hz), 7.60 (1H, dd,
Jϭ7.20 Hz), 7.52 (1H, d, Jϭ7.20 Hz), 7.39 (1H, m, Jϭ7.40, 1.20 Hz). 13C-
NMR (CDCl3) d: 157.6 (s), 156.7 (s) 140.8 (s), 139.3 (s), 137.8 (s), 137.5
(s), 135.3 (s), 135.1 (s), 129.5 (d), 126.8 (d), 124.3 (d), 122.8 (d), 116.5 (d),
116.1 (s). MS m/z: 320 (Mϩ1). Anal. Calcd for C14H6ClNO2S2: C, 52.58; H,
1.89; N, 4.38. Found: C, 52.72; H, 1.97; N, 4.19.
N-[3-(Dimethylamino)propyl]-6-oxo-5,6-dihydrothieno[3؅,2؅:4,5]-
thieno[2,3-c]quinoline-9-carboxamide Hydrochloride (10a) Method C:
A solution of 9 (0.21 g, 0.7 mmol) and [3-(dimethylamino)propyl]amine
(2.5 ml, 19.8 mmol) in toluene (20 ml) was refluxed for 30 min. After cool-
ing, the precipitate was filtered off and washed with chloroform. Recrystal-
lization from DMSO–water mixture (1 : 2, v/v), followed by conversion into
the hydrochloride salt by stirring it with saturated ethanolic HCl for 60 h,
gave white crystals (0.18 g, 65.7%), mp 275—280 °C.
Method D: A solution of 6c (0.10 g, 0.3 mmol) in [3-(N-dimethylamino)-
propyl]amine (5 ml, 39.7 mmol) was stirred at room temperature under nitro-
gen for 96 h. The excess of amine was then removed by evaporation. The
residue after evaporation was treated with ethanol in order to remove starting
material, which was then filtered off (0.01 g). The filtrate was evaporated to
dryness under reduced pressure. Recrystallization from DMSO–water mix-
ture (1 : 2, v/v), followed by conversion into the hydrochloride salt by stir-
ring it with saturated ethanolic HCl for 60 h, gave white crystals (0.04 g,
31.6%), mp 275—280 °C. IR (KBr) cmϪ1: 2696—2956 (CH2), 1645 (CϭO).
1
UV lmax (ethanol) nm: 226, 242, 282, 318, 332, 348. H-NMR (DMSO-d6)
d: 12.17 (1H, s), 10.48 (1H, s), 9.30 (1H, t, Jϭ5.70 Hz), 9.16 (1H, s), 8.48
(1H, dd, Jϭ7.86 Hz), 7.60—7.52 (2H, m, Jϭ7.98, 6.61, 1.67 Hz), 7.37 (1H,
m, Jϭ7.34, 6.65, 1.68 Hz), 3.42 (2H, dt, Jϭ6.44 Hz), 3.19 (2H, dt,
Jϭ6.69 Hz), 2.79 (3H, s), 2.78 (3H, s), 2.02 (2H, tt, Jϭ6.62 Hz). 13C-NMR
6-Oxo-5,6-dihydrothieno[3؅,2؅:4,5]thieno[2,3-c]quinoline-9-carbox-
anilide (6d) From 5d (0.50 g, 1.2 mmol) and using the same procedure as

660
Vol. 50, No. 5
(DMSO-d6) d: 161.4 (s), 157.6 (s), 146.7 (s), 144.6 (s), 141.1 (s), 137.7 (s),
135.3 (s), 134.0 (s), 129.5 (d), 126.8 (d), 123.8 (d), 122.6 (d), 116.7 (d),
116.2 (s), 54.7 (t), 42.2 (q), 42.2 (q), 36.6 (t), 24.4 (t). MS m/z: 386 (Mϩ1;
free base). Anal. Calcd for C19H20ClN3O2S2: C, 54.08; H, 4.78; N, 9.96.
Found: C, 53.96; H, 4.71; N, 10.05.
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N-[3-(Dimethylamino)propyl]-6-oxo-5,6-dihydrothieno[3؅,2؅:4,5]-
thieno[2,3-c]quinolin-9-carboxanilide Hydrochloride (10b) The chlo-
ride 9 (0.24 g, 0.8 mmol) was converted into free base of 10b by the method
described earlier for the synthesis of 5a. Free base was then converted into
the hydrochloride salt by stirring it with saturated ethanolic HCl for 15 h.
Resulting precipitate was filtered off, washed with absolute ethanol and
dried in vacuo at 50 °C for 5 h to give 10b (0.10 g, 9.1%) as white crystals,
mp 280—282 °C. IR (KBr) cmϪ1: 2361—2923 (CH2), 1655 (CϭO), 1631
(CϭO). UV lmax (ethanol) nm: 228, 242, 286, 318, 332, 348. 1H-NMR
(DMSO-d6) d: 12.12 (1H, s), 9.84 (1H, s), 7.69—7.48 (8H, m), 7.31 (1H,
dd, Jϭ7.52 Hz), 6.91 (1H, s), 3.95 (2H, t, Jϭ7.21 Hz), 3.20 (2H, t,
Jϭ8.26 Hz), 2.79 (6H, s), 1.99 (2H, tt, Jϭ8.21 Hz). 13C-NMR (DMSO-d6)
d: 161.3 (s), 157.5 (s), 147.4 (s), 143.1 (s), 141.7 (s), 140.3 (s), 137.5 (s),
134.8 (s), 134.1 (s), 130.4 (d), 130.4 (d), 129.5 (d), 129.5 (d), 129.4 (d),
129.2 (d), 124.9 (d), 122.8 (d), 122.4 (d), 116.7 (d), 115.8 (s), 54.3 (t), 47.6
(t), 42.3 (q), 42.3 (q), 22.5 (t). MS m/z: 462 (Mϩ1; free base). Anal. Calcd
for C25H24ClN3O2S2: C, 60.29; H, 4.86; N, 8.44. Found: C, 59.99; H, 5.05;
N, 8.24.
Cell Lines and Culturing Human tumor cell lines (HeLa, cervical car-
cinoma; MCF-7, breast carcinoma; CaCo-2, colon carcinoma; Mia PaCa-2,
pancreatic carcinoma; HBL, melanoma; Hep-2, laryngeal carcinoma) and
normal human fibroblasts (WI-38) were tested for sensitivity on quinolones
in vitro. All cell lines were grown in DMEM medium (supplemented with
10% heat inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml peni-
cillin and 100 mg/ml streptomycin) at 37 °C in a humidified atmosphere with
5% CO2. For the purpose of the experiment, the cells were plated in quadrip-
licate in 96-microwell flat bottom plates at the concentrations of 2ϫ104
cells/ml (all tumor cell lines) and 3ϫ104 cells/ml (WI-38) The next day
(24 h later) compounds were added to the cells at different concentrations
(10Ϫ4, 10Ϫ5, 10Ϫ6, 10Ϫ7 M). Compounds were dissolved in DMSO at the con-
centration 10Ϫ1 M and diluted with DMEM medium into working concentra-
tions. The concentration of DMSO was too small to affect the growth. Con-
trol cells (without any compound) were grown under the same conditions.
Cell viability was measured immediately before (day 0) and 72 h after ad-
dition of compounds, using MTT assay, which detects dehydrogenase activ-
ity in viable cells.32,33) For this purpose the medium was discarded and MTT
was added to each well at a concentration of 20 mg/40 ml. After 4 h of incu-
bation at 37 °C the precipitates were dissolved in 160 ml of DMSO. The ab-
sorbance was measured on ELISA reader at 570 nm, and the percentage of
growth was calculated. Each number was the mean of four parallel samples
in three individual experiments. The cytotoxic effects of each compound
were obtained as IC50 values, which represents the molar drug concentra-
tions required to cause 50% inhibition.
25) Jolivet C., Rivalle C., Bisagni E., Heterocycles, 43, 995—1005 (1996).
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28) Zhang S.-X., Feng J., Kuo S.-C., Brossi A., Hamel E., Tropsha A., Lee
K.-H., J. Med. Chem., 43, 167—176 (2000).
Acknowledgments Support of this study by the Ministry of Science
(Projects No 125005 and 00981104) and the Ministry of Small and Medium
Enterprises of Croatia is gratefully acknowledged.
29) Hour M.-J., Huang L.-J., Kuo S.-C., Xia Y., Bastow K. F., Nakanishi
Y., Hamel E., Lee K.-H., J. Med. Chem., 43, 4479—4487 (2000).
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(1990).
References and Notes
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